Aminopeptidase N1 (EtAPN1), an M1 metalloprotease of the apicomplexan parasite Eimeria tenella, participates in parasite development

Abstract

Aminopeptidases N are metalloproteases of the M1 family that have been reported in numerous apicomplexan parasites, including Plasmodium, Toxoplasma, Cryptosporidium, and Eimeria. While investigating the potency of aminopeptidases as therapeutic targets against coccidiosis, one of the most important avian diseases caused by the genus Eimeria, we identified and characterized Eimeria tenella aminopeptidase N1 (EtAPN1). Its inhibition by bestatin and amastatin, as well as its reactivation by divalent ions, is typical of zinc-dependent metalloproteases. EtAPN1 shared a similar sequence, three-dimensional structure, and substrate specificity and similar kinetic parameters with A-M1 from Plasmodium falciparum (PfA-M1), a validated target in the treatment of malaria. EtAPN1 is synthesized as a 120-kDa precursor and cleaved into 96-, 68-, and 38-kDa forms during sporulation. Further, immunolocalization assays revealed that, similar to PfA-M1, EtAPN1 is present during the intracellular life cycle stages in both the parasite cytoplasm and the parasite nucleus. The present results support the hypothesis of a conserved role between the two aminopeptidases, and we suggest that EtAPN1 might be a valuable target for anticoccidiosis drugs.

FIG 1

Bestatin inhibits E. tenella development. (A) The role of aminopeptidases in invasion (□) and development () was assayed by treatment of infected MDBK cells with bestatin. For determination of invasion, cells were fixed at 4 h p.i. and the percent invasion was calculated. For determination of development, supernatants of the cell culture were collected at 96 h p.i. and the number of first-generation merozoites was estimated and compared with that for a control without inhibitor. Values are reported as the mean ± SD of three independent experiments. Intergroup differences (P values) between the control and treated conditions were evaluated by Student's paired t test, and significance is indicated (***, P < 0.001). (B) MDBK cells infected with Wis YFP⁺ sporozoites were fixed at 72 h p.i. and mounted in Vectashield mounting medium. Cell nuclei were labeled with DAPI (blue). YFP⁺ parasites appear green. Images are representative of those from three individual experiments. Bar, 2 μm.

FIG 2

Metalloprotease activity is detected in E. tenella oocyst lysates. (A) Profile of aminopeptidase activity against H-Ala-AMC during sporulation. Protease activity from oocyst lysates was recorded at different sporulation times using H-Ala-AMC fluoropeptide. Activity was recorded after incubation of 40 μg of lysates in 10 mM phosphate buffer, pH 7.4, 70 mM NaCl at 37°C and addition of 20 μM H-Ala-AMC. Aminopeptidase activity during sporulation was normalized using the maximum activity level (observed at 6 h) as 100% peptidase activity. Intergroup differences (P values) between unsporulated (0 h) and sporulating oocysts were evaluated by Student's paired t test, and significance is indicated (*, P < 0.001). (B) Inhibition of E. tenella oocyst lysate activity by aminopeptidase inhibitors. The effect of 1,10-ortho-phenanthroline (□), bestatin (), and amastatin (■) against unsporulated oocyst lysate was assayed. The lysate was preincubated with the inhibitor before addition of the H-Ala-AMC. Values are reported as the mean ± SD of three independent experiments. Intergroup differences (P values) between the control and treated conditions were evaluated by Student's paired t test, and significance is indicated (§, P < 0.001 for 1,10-ortho-phenantroline; #, P < 0.05 for bestatin; *, P < 0.001 for amastatin).

FIG 3

Aminopeptidase activity profiling and identification of EtAPN1. The native zymography activity profile during oocyst sporulation is shown. Proteins were separated on a native acrylamide gel containing 200 μM H-Ala-AMC and observed under UV light.

FIG 4

Comparison of aminopeptidases N with Eimeria EtAPN1. Multiple-sequence alignment of the active site (boxed region) and the flanking amino acid sequence of EtAPN family members (EtAPN1, GenBank accession number KF602061; EtAPN2, ToxoDB accession number ETH_00015595 gene) and several selected members of the M1 zinc-metalloproteases (P. falciparum A-M1 [PfA-M1], GenBank accession number XP_001349846.1; E. coli APN [EcPEPN], GenBank accession number NP_415452.1; S. cerevisiae Ala/Arg aminopeptidase [ScAP2], GenBank accession number NP_011913.1; H. sapiens APN [HsAPN], GenBank accession number NP_001141.2). Identical (asterisks), 90% conserved (:), and 50% conserved (.) amino acids between all sequences are indicated. The positions of the conserved putative zinc ion ligands (Z) and the glutamyl residue required for catalytic activity (C) are indicated in bold on the bottom line. The percent amino acid identity between a given sequence and EtAPN1, in the boxed region, is indicated on the right (a). GAMEN and HEXXH motifs are in red and blue, respectively. (B) Tree of apicomplexan parasite aminopeptidases N. The tree was obtained using the MEGA (version 5) program, Muscle alignment, and the neighbor-joining method with a bootstrap of 10,000 predicted sequences from the ToxoDB database of Eimeria EtAPN2 (ToxoDB accession number ETH_00015595 gene) and Toxoplasma and Neospora aminopeptidases N (TgAPN3, ToxoDB accession number TGME49_224460; TgApN2, ToxoDB accession number TGME49_224350; TgAPN1, ToxoDB accession number TGME49_221310; NcAPN3, ToxoDB accession number NCLIV_048230; NcAPN1, ToxoDB accession number NCLIV_048240), the E. tenella N1 sequence (GenBank accession number KF602061), and sequences of Cryptosporidium and Plasmodium zinc metallopeptidases (C. hominis, GenBank accession number XP_668534.1; C. parvum A-M1 [CpAM1], GenBank accession number XP_627236.1; P. falciparum, GenBank accession number XP_001349846.1; P. vivax, GenBank accession number XP_001617047; P. berghei, GenBank accession number XP_680130.1). Human aminopeptidase N (GenBank accession number NP_01141.2) was used as an outgroup. (C) Comparison of the EtAPN1 model (red) with the X-ray crystal structures of E. coli APN (EcPEPN; PDB accession number 3B34; green) and P. falciparum A-M1 (PfA-M1; PDB accession number 3EBG; blue). Regions of structural variability are indicated by arrows. Domains are indicated i to iv.

FIG 5

Characterization of recombinant EtAPN1. Coomassie blue staining (CB), immunoblotting with anti-His antibody (α-His), and native zymography (Zym.) were performed with the 6×His-tagged purified recombinant EtAPN1. The estimated molecular mass is about 102 kDa. The recombinant protein was injected into rabbits to produce polyclonal antibodies against EtAPN1. Antibodies were assayed against the recombinant protein (α-EtAPN1r).

FIG 6

Inhibition and cation restoration of EtAPN1r activity. (A) Inhibition of EtAPN1r activity by inhibitors. Purified EtAPN1r was incubated with bestatin (□), amastatin (○), E64 (◇), and AEBSF (△) at different concentrations (1 to 100 μM) 10 min before addition of H-Ala-AMC (20 μM). The remaining activity was recorded and expressed as a percentage of the activity of the enzyme incubated without inhibitor and cations. (B) Restoration of EtAPN1r activity by metals. Purified EtAPN1r was inhibited with 1,10-ortho-phenanthroline, and the remaining activity was recorded by addition of H-Ala-AMC. To restore the activity, different concentrations (1 to 1,000 μM) of Zn²⁺ (□), Cu²⁺ (■), Co²⁺ (top ◇ curve), Mg²⁺ (○), Mn²⁺ (bottom ◇ curve), and Fe²⁺ (△) were added. Results are expressed as a percentage of the activity of the enzyme incubated without inhibitor and addition of metal ions. Values are reported as the mean ± SD of three independent experiments.

FIG 7

S1 substrate-binding pocket fingerprints of recombinant EtAPN1. The library consisted of 61 peptidyl substrates of the general structure H-aa-ACC (where the amino acids [aa] at P1 are natural and unnatural amino acids and ACC is the fluorescent leaving group). x axis, abbreviated amino acid (see Table S1 in the supplemental material for details); y axis, relative EtAPN1r activity expressed as a percentage of that of the best amino acid (i.e., homo-Phe). Values are reported as the mean ± SD of three independent experiments. dab, diaminobenzidine; Tip, (3L)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Bip, l-biphenylalanine; Bpa, 4-benzoyl-l-phenylalanine; Cba, l-2-amino-4-cyanobutyric acid; Igl, l-indanylglycine; met, methionyl; dhTrp, l-dihydrotryptophan.

FIG 8

Transcription and expression profile of EtAPN1. (A) Transcription profile of Eimeria aminopeptidase N during the life cycle. RT-PCR was performed on total RNA collected at three different times of oocyst sporulation, unsporulated (0 h), partially sporulated (12 h), and sporulated (72 h), and from second-generation merozoites (M2) and gametes (Gam). Actin was used as an internal control. (B) Cleavage pattern of EtAPN1. Lysates obtained from oocysts isolated at different times after the initiation of sporulation (0, 6, 12, 24, 48, and 72 h) and from the merozoite lysate (M2) were assessed by immunoblotting with Rα-EtAPN1r.

FIG 9

EtAPN1 transits through the nucleus within the early step of development. MDBK cells or PCKCs were infected with E. tenella Wis or Wis96 (*) strain parasites, respectively, and fixed using 2.7% PFA. MDBK cells infected with Wis strain sporozoites (Spz) were obtained at various times from 4 to 24 h p.i. Second-generation merozoites (M2) were obtained using PCKCs infected with Wis strain sporozoites. Gametes were obtained using PCKCs with Wis96 strain sporozoites (Ma, macrogamete; Mi, microgamete). Parasites were dually stained with Mα-Enolaser (Mα-Enolase_r; nucleus and surface) and Rα-EtAPN1r; cell nuclei were labeled with DAPI. Images are representative of those from three individual experiments. Bars, 5 μm.